This project is centered on the mechanisms of exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. We report two subprojects this year 1. Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells: Insulin regulates glucose transport in muscle and adipose cells through the intracellular redistribution between the cell interior and the plasma membrane (PM) of the glucose transporter 4 (GLUT4). GLUT4 content in the plasma membrane (PM) of adipose cells is determined by a dynamic equilibrium between its exocytosis and internalization. In basal adipose cells, the content of GLUT4 in the PM remains low (5%) as GLUT4 internalizes 10 times faster than it is delivered to the PM. Insulin considerably stimulates the rate of GLUT4 exocytosis with relatively little effect on the rate of internalization. Consequently, 50% of intracellular GLUT4 is translocated to the PM upon insulin activation, providing a 10-fold increase in the amount of transporter on the cell surface. GLUT4 is carried to the PM by specialized tubulovesicular compartments (referred to here as GLUT4 vesicles) tightly packed with the transporter. Thus we applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM. In summary, we propose that GLUT4 vesicles follow common pathways of constitutive exocytosis, exploiting microtubule tracks on their way to the PM and revealing constrained release of membrane cargo. However, the probability of tethering and fusion of these vesicles to the PM is specifically sensitive to insulin. Insulin is known to stimulate constitutive exocytosis in general, though to a lesser extent than GLUT4 exocytosis. We are currently investigating molecular mechanisms providing the specificity of insulin action on the GLUT4 vesicles. 2. Line Tension and Interaction Energies of Membrane Rafts Calculated from Lipid Splay and Tilt: There are suggestions that exocytosis takes place in certain membrane microdomains. Membrane domains known as rafts are rich in cholesterol and sphingolipids, and are thought to be thicker than the surrounding membrane. If so, monolayers should elastically deform so as to avoid exposure of hydrophobic surfaces to water at the raft boundary. We calculated the energy of splay and tilt deformations necessary to avoid such hydrophobic exposure. The derived value of energy per unit length, the line tension g, depends on the elastic moduli of the raft and the surrounding membrane; it increases quadratically with the initial difference in thickness between the raft and surround; and it is reduced by differences, either positive or negative, in spontaneous curvature between the two. For zero spontaneous curvature, g is 1 pN for a monolayer height mismatch of 0.3 nm, in agreement with experimental measurement. Our model reveals conditions that could prevent rafts from forming, and a mechanism that can cause rafts to remain small. Prevention of raft formation is based on our finding that the calculated line tension is negative if the difference in spontaneous curvature for a raft and the surround is sufficiently large: rafts cannot form if g is less than 0, unless molecular interactions (ignored in the model) are strong enough to make the total line tension positive. Control of size is based on our finding that the height profile from raft to surround does not decrease monotonically, but rather exhibits a damped, oscillatory behavior. As an important consequence, the calculated energy of interaction between rafts also oscillates as it decreases with distance of separation, creating energy barriers between closely apposed rafts. The height of the primary barrier is a complex function of the spontaneous curvatures of the raft and the surround. This barrier can kinetically stabilize the rafts against merger. Our physical theory thus quantifies conditions that allow rafts to form, and further, defines the parameters that control raft merger.